I want to perform the nucleofection of CRISPR/Cas9(WT) gRNA construct in beta cell line.
1. How much construct DNA shall I use for the nucleofection.
Technique- Amaxa Nucleofector Kit- T or Kit- V.
2. Also I have a two constructs out of which one has no off target effect and other has off-target. Shall I use both the constructs in 1:1 ratio or the only one with no-off target would be better?
I have used 2:1 ratio of the gRNA to Cas9. It worked well and gave me around 50% efficiency on breast cancer cell lines.
What you mean by 'no off target' ?
I have all in one construct-gRNA+ CRISPR Cas-9 along with neo selection. So shall i use antibiotic selection pressure as well for clone selection? or there is no need to go for selection pressure because once genome has been modified it wont need to retain the gRNA -CRISPR construct after 48-72 hrs of transfection.
So post 7-10 days even if i remove the G418 selection, will that b ok ?
This is because I am working with MIN 6 cell line, which shows poor growth and colony formation from single cell clones after nucleofection. Antibiotic pressure retrival post 2 weeks may give better growth property.
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